1. Field of the Invention
The present invention relates generally to the fields of nutrition and biochemistry and cellular glutathione metabolism. More specifically, the present invention relates to measurement of levels of intracellular function of cysteine and glutathione so as to provide one measurement an individual""s capability of preventing degenerative disease and dealing with oxidative stress.
2. Description of the Related Art
It is now accepted widely that a number of human health conditions, including aging, arthritis, cancer, atherosclerosis, myocardial infarction, stroke, viral infection, pulmonary conditions, bowel disease and neurodegenerative disease, can develop or be worsened by the presence of reactive oxygen molecules, commonly referred to as free radicals. These hostile molecules are normal by-products of physiological processes and are produced by metabolism of oxygen; e.g., via cellular respiration or immune system function (killing of foreign materials), and by numerous enzymatic reactions essential for metabolism. In addition, free radicals are found commonly in the environment. Environmental sources of free radicals include smoke, ionizing radiation, air pollution, chemicals (carcinogens, many petrochemicals, biocides, dyes, solvents, cytostatic drugs, etc.), toxic heavy metals and oxidized or rancid fats. Some of the most common free radicals are super oxide, hydroxyl radical, singlet oxygen, and peroxides, including hydrogen peroxides. Certain valences of iron and copper can catalyze formation of free radicals, which, although short-lived, promote a chain reaction of radical formation, followed by a wake of altered, damaged biological molecules.
Free radicals are toxic to living organisms, causing structural damage to biological molecules. Molecular damage may result in alteration of genetic codes, disruption of cell membrane integrity, neurological disorders, endocrine imbalances, increased allergies, vascular endothelial destruction and joint degradation and inflammation.
Protection from the deleterious effects of free radicals is found in a diverse range of molecules termed antioxidants. Free radicals and their chain by-products can be neutralized and converted to less harmful products by antioxidants. Antioxidants may be enzymes (such as superoxide dismutase, catalase, glutathione peroxidase), essential nutrients (such as beta carotene, vitamins C and E, selenium) or a wide variety of endogenous compounds (such as glutathione) or dietary compounds (such as the bioflavanoids). Thus, the human body has several, natural quenchers of free radicals.
Research in humans has indicated that deficient intakes of nutrient antioxidants are associated with higher risks of cancer, cardiovascular disease, arthritis, cataracts, etc. Also, a higher intake of nutrient antioxidants is associated with lower incidence of chronic degenerative diseases. Encouraging studies indicate that intervention with antioxidant nutrient supplements may have therapeutic benefit in humans.
Laboratory analysis of antioxidant status has not become routine for a variety of reasons. Free radicals are extremely fleeting and generally are not amenable to direct measurement. By-products of free radical damage can be measured, such as malondialdehyde (MDA), thiobarbituric acid-reactive substances (TBARS) or lipid peroxides in serum or urine; however, these tests may be indicators of oxidative stress but reflect only damage to certain types of biomolecules (mostly polyunsaturated lipids and nucleic acids). Yet methods of measurement of antioxidant nutrient levels in serum or cells and activities of antioxidant enzymes in cells could identify deficient levels of specific components.
Glutathione is a preeminent cellular antioxidant substance which is abundant in cytoplasm, nuclei and in mitochondria. In addition to glutathione""s powerful antioxidant function, it serves also as a powerful anti-toxin, allowing the body to eliminate numerous xenobiotic and carcinogenic compounds. Further, glutathione is essential for cell-mediated immune functions and it is critical to the maintenance of the integrity of red blood cells. Moreover, it is recognized generally that deficiencies in the glutathione system lead to significant cellular aging, and, ultimately, cellular morbidity.
The concentration of cellular glutathione has a significant effect on antioxidant function; and nutrient limitation, exercise and oxidative stress have significant effects on cellular glutathione concentrations. Under oxidative conditions, glutathione function can be depleted considerably through conjugation to xenobiotics, and by secretion of glutathione conjugates and glutathione disulfide from the affected cells. A considerable amount of glutathione may become protein bound during severe oxidative stress. Fortunately, however, compounds such as N-acetyl-cysteine are available to increase intracellular glutathione function.
Glutathione is synthesized in a series of biochemical reactions utilizing ATP, magnesium and the three amino acids glycine, glutamate and cysteine. In general, the rate of synthesis of gamma-glutamylcysteine determines the rate of synthesis of glutathione, and the sulfhydral group of cysteine provides glutathione with its biological potency. Thus, measurement of cysteine availability is essential in determining the functional availability of glutathione and in the assessment of antioxidant function.
Assessment of cysteine and glutathione also is helpful in assessing selenium deficiency. When glutathione functions as an antioxidant, it reacts with hydrogen peroxide to form glutathione disulfide in a reaction catalyzed by glutathione peroxidase. Glutathione peroxidase requires selenium as a functional cofactor. Thus, adequate cysteine function, combined with deficient or average SPECTROX(trademark) results, is indicative of an intracellular selenium deficiency and calls for further testing.
The prior art is deficient in the lack of simple cost-effective means of assessing biochemically the levels of intracellular function of cysteine and glutathione in a human, and thus an individual""s capability of dealing with oxidative stress. The present invention fulfills this longstanding need and desire in the art.
One object of the present invention is to provide a cell cuture medium useful for determining a levels of intracellular function of glutathione in lymphocytes and for performing biochemical analysis of antioxidant function, said medium comprising a buffered, serum-free solution containing the following ingredients: a carbohydrate selected from the group consisting of glucose or a compound biologically capable of producing glucose in the cells; a biologically usable form of pantothenic acid, choline or a biologically utilizable form of a substance capable of producing choline in the cells; inorganic ions comprising chloride, phosphate, calcium, magnesium, potassium, sodium, and iron in a biologically utilizable form; L-Buthionine-[S.R.]-Sulfoximine; deionized water; and a mitogen in an amount effective to stimulate the lymphocytes being assayed; said buffered, serum-free solution having a pH from about 6.8 to 7.6; and wherein said cell culture medium is characterized by being effective to determine an levels of intracellular function of glutathione in lymphocytes, nutritional deficiencies, inadequacies, and imbalances and to analyze biochemically antioxidant function of the lymphocytes.
One embodiment of the present invention includes supplementing the cell medium of the present invention with a nutrient supplement selected from the group consisting of biological utilizable forms of amino acids and vitamins, the nutrient being tested for being omitted from or being present in limiting or inhibitory amounts in the nutrient supplement.
Another object of the present invention is to provide a method of determining a levels of intracellular function of glutathione and analyzing biochemically cellular antioxidant function in an individual, comprising the steps of: inoculating the cell culture medium of the present invention with lymphocytes from an individual to be tested; incubating the inoculated cell culture medium; and comparing the response of the lymphocytes with an average response of lymphocytes from a control group of individuals.
A further object of the present invention is to provide a cell cuture medium useful for determining the level of intracellular function of cysteine and performing biochemical analysis of antioxidant function in human lymphocytes, said medium comprising a buffered, serum-free solution containing the following ingredients: a carbohydrate selected from the group consisting of glucose or a compound biologically capable of producing glucose in the cells; a biologically utilizable form of pantothenic acid, choline or a biological usable form of a substance capable of producing choline in the cells; inorganic ions comprising chloride, phosphate, calcium, magnesium, potassium, sodium, and iron in a biologically utilizable form; cumene hydroperoxide; deionized water; N-Acetyl-L-Cysteine; and a mitogen in an amount effective to stimulate the lymphocytes being assayed; said buffered, serum-free solution having a pH from about 6.8 to 7.6, said cell culture medium characterized by being effective to determine nutritional deficiencies, inadequacies, and imbalances and to analyze biochemically antioxidant function of the lymphocytes.
One embodiment of the present invention includes supplementing the cell medium of the present invention with a nutrient supplement selected from the group consisting of biologically utilizable forms of amino acids and vitamins, the nutrient being tested for being omitted from or being present in limiting or inhibitory amounts in the nutrient supplement.
An additional object of the present invention is to provide a method of determining a level of intracellular function of cysteine and analyzing biochemically cellular antioxidant function in an individual comprising the steps of: inoculating the cell culture medium of the present invention with lymphocytes from an individual; incubating the inoculated cell culture medium; and comparing the response of the lymphocytes with an average response of lymphocytes from a control group of individuals.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
The present invention is directed to a method useful for the biochemical analysis of levels of intracellular function of cysteine and glutathione in human lymphocytes. Such an analysis reflects the physiological health of an individual""s antioxidant systems within the peripheral lymphocytes. The methodology of the present invention allows precise assessment of the intracellular function of cysteine and glutathione in human lymphocytes, so that therapeutic measures can be taken to improve an individual""s antioxidant profile.
The present method, utilizing lymphocytes, offers distinct advantages, as lymphocytes: (1) are host to the cell-mediated immune system and are easily stimulated to grow (mitogenesis); (2) reflect time-averaged, long-term nutrient status (the life of a lymphocyte is about six months); (3) possess metabolic pathways common to other cells, and (4) are easily collected by standard venipuncture.
By measuring lymphocyte growth to assess intracellular function of cysteine and glutathione, the method of the present invention reflects the unique profile of each patient, which varies widely. Therapeutic treatment, therefore, can be tailored to the specific biochemical requirements of the individual rather than the xe2x80x9caveragexe2x80x9d patient as determined by so-called norms.
It will be apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion: